GSM6729210: CDC65exvivo_2; Homo sapiens; RNA-Seq
Culture protocol: Cartilage tissues were transferred into a hypoxia chamber (4% O2) immediately after being removed from OA patients and minced finely. Collagenase II at concentrations of 0.1%, , 1%, and 2% was applied for 4 or 18 hours to digest the minced tissue. Actinomycin D (ActD) was added to test its capacity in stabilizing the transcriptome. Cell yield, viability, cell size, and transcriptome were determined using counter chamber, flow cytometry, and RNA sequencing (RNA-seq). Collagenase II at 2% concentration released small chondrocytes from cartilage matrix during the first digestion hour and started to release large cells thereafter, reaching a complete release at 4 hours. During 4-hour digestions, collagenase II at 2% and 1% but not at lower concentrations yielded maximal release also of the large chondrocyte population. RNA-seq analysis, using RNA isoalted directly from the same donor as reference, revealed that a 4-hour digestion period with 1% or 2% collagenase II plus ActD optimally preserved the transcriptome. Thus, this study provides an isolation protocol for single chondrocytes from human articular cartilage optimized for transcriptome preservation and RNA-seq analysis. RNA was isolated using RNeasy Plus Micro Kit (QIAGEN). RNA quality was determined on a Bioanalyzer using the RNA 6000 Pico Kit (Agilent). Illumina libraries were prepared using Smart-Seq v4 mRNA Ultra Low Input RNA Kit (Clontech) and Nextera XT DNA Sample Preparation Kit (Illumina), with 10 ng of purified cDNA, according to the manufacturer's instructions